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An Efficient, Counter-Selection-Based Method for Prophage Curing In viruses Article An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains Esther Shmidov 1,2, Itzhak Zander 1,2, Ilana Lebenthal-Loinger 1, Sarit Karako-Lampert 3 , Sivan Shoshani 1,2 and Ehud Banin 1,2,* 1 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; [email protected] (E.S.); [email protected] (I.Z.); [email protected] (I.L.-L.); [email protected] (S.S.) 2 The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 5290002, Israel 3 Scientific Equipment Center, The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat Gan 5290002, Israel; [email protected] * Correspondence: [email protected] Abstract: Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker’s temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage- Citation: Shmidov, E.; Zander, I.; cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing Lebenthal-Loinger, I.; Karako-Lampert, S.; Shoshani, S.; Banin, E. An Efficient, with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted Counter-Selection-Based Method for in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 Prophage Curing in Pseudomonas and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this aeruginosa Strains. Viruses 2021, 13, method can support the research and growing interest in prophage biology in P. aeruginosa as well as 336. https://doi.org/10.3390/ additional Gram-negative bacteria. v13020336 Keywords: Pseudomonas aeruginosa; prophages; bacteriophages; curing; counter-selection; lysogen Academic Editor: Dann Turner Received: 2 February 2021 Accepted: 18 February 2021 1. Introduction Published: 21 February 2021 Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium with a single flagel- lum. It is an opportunistic pathogen of plants, nematodes, insects, animals, and humans [1]. Publisher’s Note: MDPI stays neutral It can cause a wide range of acute and chronic infections, such as in severe wounds or with regard to jurisdictional claims in published maps and institutional affil- burns, and chronic lung infections in cystic fibrosis (CF) patients [2,3]. This is enhanced iations. by the bacterium’s low susceptibility to various antimicrobial substances, making most of the infections difficult to treat and life threatening [4,5]. The genetic variability among P. aeruginosa isolates is relatively low; different P. aeruginosa strains share a conserved “core genome” and differ in the variable “accessory segments” [6]. The accessory genome in P. aeruginosa consists mainly of genomic islands and mobile genetic elements (MGEs) such Copyright: © 2021 by the authors. as plasmids, transposons, and prophages-like elements (temperate bacteriophages). Licensee MDPI, Basel, Switzerland. Bacteriophages (phages) are viruses that infect bacteria. They are highly abundant, This article is an open access article distributed under the terms and rapidly spreading, and very diverse biological entities. Phage infections divide into pro- conditions of the Creative Commons ductive lytic or lysogenic (temperate), depending on several factors such as host genetics, Attribution (CC BY) license (https:// phage genetics, environmental conditions, and phage concentration [7]. In the lytic life creativecommons.org/licenses/by/ cycle, phage infection is usually followed by intensive viral DNA replication, eventually 4.0/). leading to a high rate of new phage production and bacterial host cell lysis. In the lysogenic Viruses 2021, 13, 336. https://doi.org/10.3390/v13020336 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 336 2 of 12 life cycle, the viral genome is integrated into the bacterial genome as a prophage and further replicates throughout bacterial cell division. A temperate phage can be triggered into the lytic cycle (excision event) spontaneously at a low frequency or by an external stressor such as bacterial DNA damage and physical or chemical factors [7]. The integration of a lysogenic phage into the bacterial genome can influence bacterial phenotypes by providing new traits such as antibiotic resistance, metabolic factors, as well as immunity against specific viral re-infections [7,8]. In some pathogenic bacterial species, prophages encode virulence factors and mediate bacterial adaptation in ecological niches [9]. As mobile elements, prophages are an essential tool for horizontal gene transfer between bacteria and, among other factors, contribute to strain genetic variability. Most P. aeruginosa strains were identified to contain at least one prophage-like element; some are poly-lysogens, harboring several prophages in their genome [10,11]. Lysogenic phages in P. aeruginosa have been shown to confer selective beneficial traits, such as O antigen conversion, biofilm development, and virulence [12–14]. The PAO1 laboratory strain contains Pf1-like filamentous phage (Pf4) as a prophage that plays a role in small colony variant (SCV) formation during P. aeruginosa biofilm development and has a symbiotic role in bacterial biofilm formation [15,16]. Moreover, 39016 is a clinical isolate of P. aeruginosa that phenotypically differs from the laboratory wild-type (WT) strain PAO1; it exhibits a slower growth rate, reduced motility, and higher biofilm production levels [17]. The prophage-region screening of the published genome of the 39016 strain revealed that it contains five different prophage-like regions in its genome, and four of them are predicted to be intact and able to create infectious virions. As 39016 and PAO1 share the high conserved "core genome" and considering the phenotypic variation among these two strains, the prophage regions of 39016 are likely to influence the bacterial physiology and virulence. A comparison of isogenic strains that differ only in their prophage region can con- tribute to understanding the biological role of a specific phage to the observed traits, the overall prophages contribution, and the cross-regulation between the prophages in poly- lysogenic strains. The main difficulty for this type of study is the lack of a simple and efficient experimental method for curing prophages in P. aeruginosa strains. Previously described prophage curing methods, both for Gram-negative and Gram- positive bacteria, mostly relied on promoting the phage loss with the use of DNA-damaging reagents and the activation of the SOS-response and thus potentially causing a variety of genomic mutations in the bacterial genome, which might result in further variation of the phenotypes [18,19]. Here, we introduce a simple, non-SOS based, highly efficient prophage curing method in P. aeruginosa, using the Gram-negative popular counter-selection marker sacB [20]. We applied the prophage curing method on Pf4 from the WT strain PAO1 and a prophage from the clinical isolate 39016, herein termed PR2. The method can theoretically be applied for all the Gram-negative bacteria containing an intact, excisable prophage in their genome, allowing a better investigation of the prophages and their contribution to bacterial physiology and virulence. 2. Materials and Methods 2.1. Bacterial Strains, Plasmids and Growth Media The bacterial strains and plasmids used in this study are listed in Table1. Primers are listed in Supplementary Table S1. All strains were grown on LB (Luria-Bertani broth, Difco, Sparks, MD, USA) at 37 ◦C unless otherwise specified. For the insertion processes, the following media were used: Vogel Bonner Minimal Medium (VBMM) [21], Pseudomonas Isolation Agar (PIA, Difco, Sparks, MD, USA), and No salt Luria-Bertani (NSLB) + 10% sucrose. For the plasmid curing assay and DH5α heat shock, BHI (Brain Heart Infusion broth, Difco, Sparks, MD, USA) media was used. Antibiotic concentrations used in this study were 300 µg/mL carbenicillin (Crb) and 50 µg/mL gentamicin (Gm) for P. aeruginosa and 100 µg/mL ampicillin (Amp) and 30 µg/mL Gm for Escherichia coli. Viruses 2021, 13, 336 3 of 12 Table 1. Strains and plasmids used in the study. Strain or plasmid Description Source P. aeruginosa strains PA01 WT [22] PAO1 DPf4 PAO1, DPf4 This study PAO1 DpfiT PAO1, DPAO729::Crbr [23] LMG 27,647 P. aeruginosa (Schoeter 1872) BCCM-biological origin: 39016 migula 1900 AL keratitis patients 39016/PR2_ampR 39016, DPA39016_000100015::Crbr This study 30916 DPR2/Gmr 39016, DPR2::Gmr This study 30916 DPR2 39016, DPR2 This study E. coli strains F- F80lacZDM15 D(lacZYA-argF) U169 DH5α recA1 endA1 hsdR17 (rK-, mK+) phoA Bio-Lab supE44 λ- thi-1 gyrA96 relA1. E. coli S17 thi, pro, hsdR, recA::RP4 S17 [24] -2-Tc::Mu aphA::Tn7, λ-pir, Smr, Tpr Plasmids
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